Characterization of antibiotic resistance genes and mobile genetic elements in Escherichia coli isolated from captive black bears

The objective of this study was to analyze the antimicrobial resistance (AMR) characteristics produced by antibiotic resistance genes (ARGs), mobile genetic elements (MGEs) and gene cassettes in Escherichia coli isolated from the feces of captive black bears. Antimicrobial susceptibility testing was performed by using the disk diffusion method, and both MGEs and integron gene cassettes were detected by polymerase chain reaction. Our results showed that 43.7% (62/142) of the isolates were multidrug resistant strains and 97.9% (139/142) of the isolates were resistant to at least one antibiotic. The highest AMR phenotype was observed for tetracycline (79.6%, 113/142), followed by ampicillin (50.0%, 71/142), trimethoprim-sulfamethoxazole (43.7%, 62/142) and cefotaxime (35.9%, 51/142). However, all isolates were susceptible to tobramycin. tetA had the highest occurrence in 6 ARGs in 142 E. coli isolates (76.8%, 109/142). Ten mobile genetic elements were observed and IS26 was dominant (88.0%, 125/142). ISECP1 was positively associated with five β-lactam antibiotics. ISCR3/14, IS1133 and intI3 were not detected. Seventy-five E. coli isolates (65 intI1-positive isolates, 2 intI2-positive isolates and 8 intI1 + intI2-positive isolates) carried integrons. Five gene cassettes (dfrA1, aadA2, dfrA17-aadA5, aadA2-dfrA12 and dfrA1-aadA1) were identified in the intI1-positive isolates and 2 gene cassettes (dfrA1-catB2-sat2-aadA1 and dfrA1-catB2-sat1-aadA1) were observed in the intI2-positive isolates. Monitoring of ARGs, MGEs and gene cassettes is important to understand the prevalence of AMR, which may help to introduce measures to prevent and control of AMR in E. coli for captive black bears.

Antimicrobial resistance (AMR) is a rapidly growing global health problem in animals and humans 1 .Antimicrobial resistance genes (ARGs) have been considered as a major mechanism of bacterial resistance to antibiotics 2 .Furthermore, the presence of bacteria carrying ARGs has been widely reported in wildlife in previous studies, which are major factors in the emergence of global health challenges 3 .Mobile genetic elements (MGEs), such as integrons, transposons and plasmids, play an important role in the spread of ARGs in animals and the environment 4 .Integrons, a type of MGE, can easily transfer one or more gene cassettes between different bacteria.Different gene cassettes that are incorporated into integrons have a significant correlation with resistance to different antibiotics 5,6 .
Black bear (Ursus thibetanus) is one of the national first-class protected wild animals in China, and there are more than 97 facilities are maintained for black bears and other bears in China 7,8 .There have been more reports on the epidemiological investigation of antibiotic resistant bacteria in captive wild animals [9][10][11] ,and study for E. coli from sloth bear in India showed high prevalence of antimicrobial resistance, indicating that the AMR E. coli from captive black bears should be concerned 12 .Overuse of antibiotics has led to a problem of antibiotic resistant in clinical practice 13 .Previous studies have shown that the feces of healthy wild animals may serve as a reservoir for antibiotic resistant Escherichia coli (E.coli), which could pose a threat to public health and environmental

Statistical analysis
All positive PCR products were directly sequenced in both directions by BGI (Beijing, China).Sequences were analyzed online by BLAST (http:// blast.ncbi.nlm.nih.gov).P-values < 0.05 were considered to be statistically significant.The association between AMR phenotypes and the MGEs was calculated, and was considered significant at a P-value of < 0.05 and was reported as an odds ratio (OR) with 95% confidence interval (CI).An OR > 1 was considered a positive association or an increasing likelihood of co-occurrence of the MGEs or AMR phenotype, while an OR < 1 was considered a negative association.The statistical analyses were performed using the SPSS 27 software (StataCorp Lp, College Station, TX, USA).

Ethics approval
This study was reviewed and approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University under permit number DYY-2020103018.Prior to the collection of fecal specimens from captive black bear, permission was obtained from the farm of black bear breeding, Dujiang Yan city, China.

Discussion
To enhance comprehension of the AMR of E. coli isolates from captive black bears, we assessed 142 E. coli strains from 142 captive black bears.Of these strains, 43.7% exhibited multidrug resistance (MDR).The MDR rate of E. coli in black bears is similar to that of sloth bear (51.1%) and giant panda (43.4%) 24,25 , but lower than that of domestic animals and poultry 26 .Antibiotics have been widely applied to promote growth and prevent diseases in China, which may lead to high MDR strains detected in domestic animals and captive wild animals [27][28][29] .Our study found a high rate of antibiotic resistance in E. coli isolates (97.9%) from black bears, which is higher than that observed in sloth bears in India (93.3%) 25 .The consumption and production of antibiotics are higher in China 30 , and the frequent exposure or misuse of antimicrobials in animals may contribute to the emergence and spread of resistance in E. coli from black bears.In our study, E. coli from black bear exhibited varying degrees of resistance to GM, CIP, CTX and CXM.GM, CIP, CTX and CXM have been used for disease control in black bears, which may contribute to antimicrobial resistance.Additionally, the resistance rate to TE (79.6%) in E. coli is the highest rate among 13 antibiotics 31 .The high resistance rate to TE (79.6%) has also been reported in sloth bears (51.1%) 25 .TE has been used to treat animal infections in the world 32 , however, the development of resistance has narrowed their utility and the use of TE is strictly regulated in China 33 .Our previous study was also found this phenomenon in giant panda, indicating that AMR persists for a longer time and is not easy to eliminate 34 .In general, ARGs were the primary cause of antibiotic resistance phenotypes, AGRs were expressed and translated into proteins, allowing bacteria to achieve resistance to antibiotics.Different ARGs mediate bacterial resistance to antibiotics in different ways 35 .In our study, 6 ARGs were detected and the most prevalent ARG was tetA (70.8%, 109/142).The high prevalence of tetA has also been observed in various wild animals (e.g., giant panda, monkey) and human studies 23,24,36 , suggesting that TE resistance will continue to exist for a period of time, and we should continue to monitor TE resistance.Moreover, we also found that some isolates (DJY62 and DJY129) showed antimicrobial susceptibility despite harboring ARGs.This phenomenon may be related to abnormal expression of ARGs, or the expression of ARGs may not have reached the level required to produce resistance 37 .
Previous studies have shown that MGEs play an important role in the dissemination of ARGs 38,39 .Our result showed that 10 MGEs and 23 MGEs combination patterns were detected in 142 E. coli strains from black bears.In comparison to our previous research, we found fewer MGEs (10) and MGEs combinations (23) in black bears than in giant pandas (11 MGEs, 35 MGEs combinations) 24 .We detected 10 bla CTX-M positive strains out of 13 strains carrying ISECP1 (Table S2).Studies have shown that ISECP1 and bla CTX-M are all located in the same plasmid, which may be the reason for their association with five β-lactams antibiotics 40 .Horizontal transfer of ARGs mediated by MGEs is one of the main mechanisms 41,42 , and it is necessary to regularly monitor MGEs in E. coli from black bears.To our knowledge, this study is the first to examine MGEs of E. coli isolates from captive black bears.
Integrons, as one of the MGEs, are known to capture gene cassettes, which could be transferable among bacteria and disseminate ARGs via transmissible plasmids and insertion sequences, posing a threat to public health 16,43 .In our results, class 1 integron (56.8%) was more prevalent compared to class 2 integron (6.3%),Table 3.The associations between MGEs and AMR among E. coli isolates from black bear in Sichuan, China (n = 142).*Only significant association (P < 0.05) are shown.µ Odds ratio (OR) for significant associations between MGEs and AMR (95% confidence interval in parenthesis); NA, no results available (or) could not be calculated because none of the isolates carried one of the pairs of MGEs and AMR or one of the rate of detected was zero).-indicates no significant associations (P > 0.05).www.nature.com/scientificreports/while class 3 integron was not detected, which is consistent with results from yaks, ducks and giant pandas 16,24,44 .
Research has shown that due to the influence of human activities, the prevalence of Class I integron in natural environments is higher than that of Class II and III 45 .Gene cassettes harbored in integron-positive isolates are an important medium for the spread of ARGs 46 .In this study, seven types of gene cassettes were detected in class 1 or class 2 integron-positive isolates (Table 2).The gene cassette encoding the aminoglycoside adenyl transferase (aadA2) and dihydrofolate reductase (dfrA12) 34 is the most frequently detected among the isolates, with 11 instances 34 .Similar gene cassettes belonging to dfrA, sat, catB and aadA have also been detected in E. coli isolates from other animals, including giant pandas and rabbits 24,47 , indicating that the gene cassettes have spread between different species.
The above results indicate that different types of integrons and gene cassettes are carried by E. coli from captive black bears, posing a threat of spreading ARGs to other animals or environments.The high prevalence of AMR E. coli detected in black bears pose a potential risk to public health.Therefore, relevant people like veterinarians should pay attention during work.The origin of the AMR isolates analyzed in our present study is unclear, despite the high prevalence of AMR E. coli detected within the farm-raised black bear population.

Conclusion
This study found a high prevalence of antibiotic resistance and a diverse range of MGEs observed among the E. coli strains isolated from captive black bears.A positive association was observed between MGEs and AMR in E. coli strains.It is essential to monitor the distribution of ARGs, MGEs and gene cassettes, which may help to introduce interventions for the prevention and control of AMR in E. coli among captive black bears.

Figure 1 .
Figure 1.E. coli from black bears feces samples was resistant to various antibiotics.The highest resistance rate was TE (79.6%), followed by AMP (50.0%) and SXT (43.7%).All strains are sensitive to TOB.

Figure 2 .
Figure 2. The resistant phenotype patterns of E. coli isolates from black bears.(A) The 0 on the x-axis means the strains were sensitive to all antibiotics tested and 1-5 means the strains resistant to 1-5 categories of antibiotics, respectively.The bars indicating that 62 E. coli isolates (62/142, 43.7%) are MDR.(B) Color bars demonstrating the distribution of resistant phenotype patterns in E. coli isolates from black bears and only MDR isolates were analyzed (n = 62).By using disk diffusion assay, a total of 40 resistant patterns were observed in MDR E. coli strains.

Figure 3 .
Figure 3. Components of mobile genetic elements in 142 E. coli isolates from black bears.Color bars demonstrating the distribution of MGEs combination patterns in E. coli isolates from black bears, a total of 23 MGEs combination patterns were observed in E. coli strains.

Table 1 .
Distribution of ARGs and AMR detected in 142 E. coli strains.

Table 2 .
Gene cassettes among 72 integron positive E. coli isolates from black bear."-": Gene cassettes not detected in this study.